Method of destroying and preventing bacterial and fungal biofilm by amino acid infusion

ABSTRACT

Disclosed is a method comprising the administration of a 3% amino acid and 3% glycerin solution for the use of prevention and disruption of bacterial biofilms. Also disclosed is method comprising the administration of L-cysteine 0.4 g per 100 ml for the prevention and destruction of fungal biofilms.

CROSS-REFERENCE TO RELATED APPLICATION

This application is related to Non-Provisional Patent Application13/912,060, filed Jun. 6, 2013, titled “A Method Of Destroying BacterialBiofilm Using Sterile Intravenous Or Intracavernous Glycerin” and toNon-Provisional Patent Application Ser. No. 13/373,445, filed Nov. 15,2011, titled “Method of Treating a systemic inflammatory disorder anddamaged internal tissues.”

FIELD OF INVENTION

This invention pertains to the use of solutions administered in theinhibition and destruction of bacterial and fungal biofilms.

Description of Related Art

A biofilm occurs when microbes stick to each other on a surface. Theseadherent microbial cells are frequently embedded within a self-producingmatrix of extracellular polymeric substance. Biofilms are also referredto as slime. The polymeric conglomeration is generally composed ofextracellular DNA, proteins and polysaccharides. Initially the biofilmis weak and adhesion is by van der Waals forces. Later, the microbesform cell adhesion structures such as pili in the case of bacteria orhyphae in the case of fungi. Once colonization has begun, the biofilmgrows through a combination of cell division and recruitment ofextracellular components.

The development of a biofilm may allow for an aggregated cell colony tobe increasingly antibiotic resistant. Microbes from the biofilm candisperse which causes the spread and colonization of new surfaces. Theextracellular matrix protects the microorganisms within it andfacilitates communication among them through biochemical signals.Biofilms have been implicated in such problems as urinary tractinfections, endocarditis, cystic fibrosis and infections of medicaldevices, such as prostheses and heart valves. Invariably the onlyrecourse for treating prosthetic devices such as mechanical heart valvesis to have them replaced. Biofilms are present on the removed tissue of80% of patients undergoing surgery for chronic sinusitis.

Danish pioneers first connected biofilms with human disease in the1980′s and then with antibiotic resistant infections. They discoveredthat once these biofilm infections had begun they are difficult to getrid of in the body. The immune system can mop up free-floating microbesin the blood but reaching bacteria and fungi within the biofilmreservoir is difficult.

Even if an antimicrobial agent reaches a biofilm, a large portion of themicrobes would be insensitive to the specific antimicrobial agent asbacteria and fungi in a biofilm typically exist in a dormant state. Thedormant microbes are not vulnerable to the antimicrobial agent. Later,these dormant microbes can quickly renew the biofilm. Low oxygenconcentrations in the biofilm also protects the microbes from someantimicrobial agents, which require aerobic metabolism.

According to the Center for Disease Control, 65% of treated bacterialinfections develop from a biofilm. Biofilms are implicated in chronicinfections. Most notable among them is Staphylococcus aureus, especiallythe methicillin resistant (MRSA) variety. Also, an estimated 13% ofintensive care patients have a fungal infection likely originating froma biofilm.

SUMMARY OF INVENTION

Disclosed is a method comprising the administration of a 3% amino acidand 3% glycerin solution for the use of prevention and disruption ofbacterial biofilms.

Also disclosed is method comprising the administration of L-cysteine 0.4g per 100 ml for the prevention and destruction of fungal biofilms.

DETAILED DESCRIPTION

A 66 year old female developed cellulitis and an open ulcer on her leg.This was treated by oral antibiotics and was also treated at anoutpatient wound care center. When the outpatient care was notsuccessful she was admitted to the hospital for intravenous antibiotics.After 3 weeks there was no sign of improvement and the ulcer wasenlarging. At this stage the ulcer measured 3 centimeters by 1½centimeters in size and there was surrounding redness suggestive ofinflammation. She was then given an intravenous solution of 3% aminoacids and 3% glycerin at an infusion rate of 80 cubic centimeters (cc)per hour. At the end of 48 hours there was evidence of healing in theulcer. During these 48 hours, the patient was continued on intravenousantibiotics. After 72 hours the patient was discharged home on oralantibiotics. When she was re-examined 3 weeks later, there was noevidence of the ulcer and the surrounding inflammation that was causedby cellulitis had totally cleared. Ulcers such as this suggest that thepatient had developed a bacterial biofilm that had increased resistanceto antibiotics causing the antibiotics to be ineffective.

In the lab we discovered that glycerin had little effect on biofilminhibition.

ProcalAmine® which contains the amino acids Isoleucine 0.21 g per 100ml, Leucine 0.27 g per 100 ml, Lysine (as Lysine Acetate USP 0.31 g)0.22 g per 100 ml, Methionine 0.16 g per 100 ml, Phenylalanine 0.17 gper 100 ml, Tryptophan 0.046 g per 100 ml, Valine 0.2 g per 100 ml,Alanine 0.21 g per 100 ml, Arginine 0.29 g per 100 ml, Histidine 0.085 gper 100 ml, Proline 0.34 g per 100 ml, Serine 0.18 g per 100 ml, Glycine0.42 g per 100 ml, Threonine 0.12 g per 100 ml, Cysteine (as L-cysteinehydrochloride monohydrate less than 0.020 g) less than 0.014 g per 100ml, both inhibited and destroyed bacterial and fungal biofilms.

We found that Aminosyn 10% which contained no L-cysteine and alsocontained Tyrosine, Aspartic Acid, and glutamic acid did not inhibit ordestroy bacterial and fungal bio films. It was not aided by the additionof L-cysteine for the bacterial bio films, but was aided by the additionof L-cysteine for the fungal biofilms. This may indicate that tyrosine,aspartic acid, or glutamic acid may inhibit the destructive effect ofone or some of the other amino acids on the bacterial biofilm.

We also discovered that L-cysteine alone at a concentration of 0.4 g per100 ml caused inhibition and destruction of fungal biofilms, such asthose formed by the predominant human fungal pathogen, Candida albicans.L-cysteine alone had no effect on the bacterial biofilms.

The method used was as follows:

Inhibition and Disruption Assay In Vitro

Candida albicans wild-type strain SN250 was incubated on YEPD plates at30° C. at 225 rpm for 16 hrs. Staphylococcus aureus wild-type strain JE2was incubated on Blood Agar plates at 37° C. for 24 hours. A singlecolony from the plate was inoculated in 4 mL of TSB media and incubatedat 37° C. on a rotating platform for 16 hours.

Biofilms were grown as follows: For the inhibition assay, the cells wereincubated in RPMI-1640 medium alone and with compounds to be tested (1%ProcalAmine® or 0.4% L-cysteine) at 37° C. at 250 rpm for C. albicansand statically for S. aureus for 90 minutes, for cell adhesion. Cellswere washed with PBS solution followed by addition of RPMI-1640 aloneand with compounds to be tested (1% ProcalAmine® or 0.4% L-cysteine),and further incubated at 37° C. at 250 rpm (for C. albicans) andstatically (for S. aureus) for 24 hours for biofilm growth.

For the disruption assay, the biofilm was grown in RPMI-1640 alone,following the same procedure mentioned above for C. albicans and S.aureus, with no addition to media. After 24 hours, the media wasaspirated and carefully replaced by RPMI-1640 alone and with treatments(1% ProcalAmine® or 0.4% L-cysteine) and further incubated at 37° C. at250 rpm (C. albicans) and statically (S. aureus) for 24 hours.

All biofilms formed were analyzed by measuring optical density at 630nm. Six replicates for each treatment were performed.

These same amino acids may be used in both the prevention and thetreatment of biofilms in human and animal bacterial biofilms andL-cysteine may be used in the treatment and prevention of fungal biofilmin both humans and animals.

Consequently, these solutions may be used for oral and topicalprevention and treatment.

In some embodiments, a 3% amino acids and 3% glycerin solution (e.g.,P_(roca)lA_(m)i_(ne)®) may be used for oral and topical prevention andtreatment with a swish and swallow method, repeated as needed. In someembodiments, the 3% amino acids and 3% glycerin solution may be used asa topical method of treatment through the irrigation of a wound with the3% amino acids and 3% glycerin solution followed by dressing the woundwith a compress moistened with the 3% amino acids and 3% glycerinsolution and maintained for approximately 48 hours or due to differencesin wound area and depth, until satisfactory treatment.

In some embodiments, a 0.4% L-cysteine solution may be used for oral andtopical prevention and treatment of C. albicans biofilms with a swishand swallow method, and repeated as needed. In some embodiments, a 0.4%L-cysteine solution may be used with as a topical method for theprevention and treatment of C. albicans biofilm infections through theirrigation of a wound with 0.4% L-cysteine solution followed by dressingthe wound with a compress moistened with 0.4% L-cysteine solution andmaintained for approximately 48 hours, or due to differences in woundarea and depth, until satisfactory treatment.

In some embodiments, a 3% amino acids and 3% glycerin solution (e.g.,P_(roca)lA_(m)i_(ne)®) may be used for the treatment of biofilmsindicated in chronic sinusitis. In some embodiments, a 0.4% L-cysteinesolution may be used for the treatment of biofilms indicated in chronicsinusitis.

1. A method of preventing formation of and/or destroying a bacterialbiofilm comprising administering a solution of 3% glycerin and a totalof 3% amino acids, wherein the step of administering comprisesirrigating a wound with the solution, nasal irrigation with thesolution, orally swishing and spitting, intravenous infusion, orapplying a compress moistened with the solution to the wound.
 2. Themethod of claim 1, wherein the solution comprises a total isoleucine at0.21 g per 100 ml, Leucine at 0.27 g per 100 ml, Lysine at 0.22 g per100 ml, Methionine at 0.16 g per 100 ml, Phenylalanine at 0.17 g per 100ml, Tryptophan at 0.046 g per 100 ml, Valine at 0.2 g per 100 ml,Alanine at 0.21 g per 100 ml, Arginine at 0.29 g per 100 ml, Histidineat 0.085 g per 100 ml, Proline at 0.34 g per 100 ml, Serine at 0.18 gper 100 ml, Glycine at 0.42 g per 100 ml, Threonine at 0.12 g per 100ml, and L-cysteine at 0.014 g or less per 100 ml.
 3. The method of claim1, wherein the solution is ProcalAmine®.
 4. The method of claim 1,wherein the step of administering comprises applying a compressmoistened with the solution to the wound for 48 hours.
 5. The method ofclaim 1, wherein the method of administration is by nasally irrigating awound with the solution of 3% amino acids and 3% glycerin.
 6. The methodof claim 1, wherein the biofilm comprises Staphylacoccus aureus.
 7. Themethod of claim 1, wherein the method of administration is swishing andspitting the solution orally.
 8. A method of treating a fungal biofilmin an animal comprising administering a solution comprising L-cysteine.9. The method of claim 8, wherein the step of administration isperformed intravenously, topically, orally, or intranasally.
 10. Themethod of claim 8, wherein the composition further comprises asparticacid and/or glutamic acid.
 11. The method of claim 8, wherein thecomposition further comprises one or a combination of: isoleucine,leucine, lysine, methionine, threonine, valine, alanine, arginine,histidine, proline and serine.
 12. (canceled)
 13. The method of claim 8,wherein the biofilm comprises Candida albicans.
 14. The method of claim8, wherein the step of administering comprises exposing the biofilm to acompress moistened with the solution.
 15. A method of preventingformation of a fungal biofilm in an animal comprising administering tothe animal a solution comprising L-cysteine.
 16. The method of claim 15,wherein the step of administration is performed intravenously, topicallyor orally.
 17. The method of claim 15, wherein the composition furthercomprises aspartic acid and/or glutamic acid.
 18. The method of claim15, wherein the composition further comprises one or a combination of:isoleucine, leucine, lysine, methionine, threonine, valine, alanine,arginine, histidine, proline and serine.
 19. (canceled)
 20. The methodof claim 15, wherein the biofilm comprises Candida albicans.
 21. Themethod of claim 15, wherein the step of administering comprises exposingthe solution to the biofilm orally by swishing and splitting.
 22. Amethod of treating or preventing sinusitis in an animal subjectcomprising administering to the animal a composition comprisingProcalAmine®.
 23. The method of claim 22, wherein the animal is a human.